This proposal is to finish the sequence of the entire chromosome of the bacterium E. coli. The sequence will be determined using the Sanger dideoxy method implemented on AB fluorescence sequencing machines. The strategy chosen entails an initial phase of random shotgun sequencing followed by reverse strand sequencing by the Janus vector and finally primer walking to achieve closure. A novel method is proposed for isolating suitable fragments of the genome to avoid resequencing already sequenced portions. These fragments will be defined by pairs of unique specific endonuclease cut sites introduced into the genome through mini- transposon metagenesis and will be isolated in high yield through preparative pulse field gel electrophoresis. The backup strategy entails sequencing already available lambda clones of E. coli DNA. The sequence of E. coli, when completed, will provide a major resource for understanding the genetics of the bacterial cell and by extension, of the human cell, for many of the genes of human will find homologs in the bacterial sequence. The sequence will also be of value in understanding the mechanisms of bacterial pathogenesis.